Protein assays are often involved in biochemical studies and are the basis for many experiments. Therefore, it is important to understand the protein assay method. At present, there are five methods commonly used for protein determination: Kjeldahl, Biuret, Folin-Lowry, UV, and Coomassie Blue (Bradford). . Among the five assays, the Coomassie brilliant blue method (Bradford method) and the Folin-phenol reagent method (Lowry method) have the highest sensitivity, while the nitrogen determination method is the most complex but accurate. In general, we use the nitrogen determination method to measure standard proteins as reference values. Therefore, the Kjeldahl method is more commonly used in reality. Of course, we have to consider the following aspects when selecting protein assays: 1. The sensitivity and accuracy required for the assay; 2. The nature of the protein; 3. The presence of interfering substances in the solution; 4. The cost of the assay time. Below, we will take a closer look at various measurement methods and their advantages and disadvantages.

(a) Kjeldahl method:

The sample was heated with concentrated sulfuric acid. Nitrogen-containing organic matter decomposes to produce ammonia (digestion), and ammonia reacts with sulfuric acid to become ammonia sulfate. Alkalinization of strong bases allows the separation of ammonia to release ammonia by steam to the acid solution. The nitrogen content of the sample can be calculated based on the degree of neutralization of the acid solution. If glycine is taken as an example, the reaction formula is as follows:

CH2COOH

| + 3H2SO4 ® 2CO2 + 3SO2 +4H2O + NH3 (1)

NH2

2NH3 + H2SO4 ® (NH4)2SO4 (2)

(NH4)2SO4 + 2NaOH ® 2H2O +Na2SO4 + 2NH3 (3)

Reactions (1) and (2) were completed in a Kjeldahl flask, and reaction (3) was performed in a Kjeldahl distillation apparatus.

To accelerate digestion, CuSO4 can be added as a catalyst, K2SO4 to increase the boiling point of the solution. Aqueous solutions can be used for the collection of ammonia and strong acids for titration. The calculated result is the total nitrogen of the sample. If you want to obtain the protein content in the sample, you should subtract the total nitrogen from the non-protein nitrogen. To further determine the protein content in the sample, multiply the protein nitrogen in the sample by 6.25.

(B) Biuret method (Biuret method)

Principle of the experiment: Biuret (NH3CONHCONH3) is a product obtained by heating two molecules of urea at about 180°C and releasing one molecule of ammonia. In a strongly basic solution, biuret forms a purple complex with CuSO4, known as the biuret reaction. Where there are two amide groups or two directly linked peptide bonds, or peptide bonds that can be connected through an intermediate carbon atom, these compounds have a biuret reaction.

The depth of the purple complex color is directly proportional to the protein concentration, and has nothing to do with the protein molecular weight and amino acid composition, so it can be used to determine the protein content. The measurement range is 1 to 10 mg protein. Substances that interfere with this assay are: ammonium sulfate, Tris buffer, and certain amino acids.

This method is quick and easy but has poor sensitivity. When it is necessary to measure the protein content in a short time, this method can be selected.

(c) Folin-phenol reagent method (Lowry method)

This protein assay is one of the most sensitive methods. In the past, this method was the most widely used method. Due to the difficulty in formulating Reagent B (now available for ordering), it has been gradually replaced by Coomassie Blue in recent years. The color rendering principle of this method is the same as the biuret method, except that the second reagent, Folin-phenol reagent, is added to increase the amount of color development, thereby improving the sensitivity of detecting proteins. The reason why these two chromogenic reactions produce dark blue color is: Under alkaline conditions, peptide bonds in proteins combine with copper to form complexes. Phosphomolybdate-phosphotungstate in the Folin-phenol reagent is reduced by tyrosine and phenylalanine residues in the protein, producing dark blue (a mixture of molybdenum blue and tungsten blue). Under certain conditions, the depth of blue is proportional to the amount of protein.

Folin-phenol reagent method was first established by Lowry to determine the basic steps of protein concentration determination. Later in the field of biochemistry has been widely used. The advantages of this assay are its high sensitivity, which is much more sensitive than the biuret method. The disadvantage is that it takes a long time and requires precise control of the operating time. The standard curve is not a strictly linear form, and the specificity is poor, and the interference substance is relatively poor. many. Ions that interfere with the biuret reaction are also likely to interfere with the Lowry reaction. And the impact on the latter is much greater. Phenolics, citric acid, ammonium sulfate, Tris buffer, glycine, sugars, glycerol, etc. all interfere. Low concentrations of urea (0.5%), sodium sulphate (1%), sodium nitrate (1%), trichloroacetic acid (0.5%), ethanol (5%), ether (5%), acetone (0.5%), etc. The solution has no effect on the color development, but when the concentration of these substances is high, a calibration curve must be made. Ammonium sulfate solution, only add concentrated sodium carbonate - sodium hydroxide solution, can be color measurement. If the acidity of the sample is high and the color is light after the color is developed, the concentration of the sodium carbonate-sodium hydroxide solution must be increased by 1 to 2 times.

When performing the assay, be careful when adding the folin-phenol reagent because the reagent is only stable under acidic pH conditions, but the above reduction reaction only occurs at pH=10, so when the Folin-phenol reagent is added to the alkaline The copper-protein solution must be mixed immediately so that the reduction reaction can occur before the phosphomolybdic acid-phosphotungstic acid reagent is destroyed.

This method is also suitable for the quantitative determination of tyrosine and tryptophan.

This method can detect the minimum protein mass of 5mg. The usual measurement range is 20 to 250 mg.

(D) UV absorption method

In the protein molecule, the benzene rings of tyrosine, phenylalanine and tryptophan residues contain conjugated double bonds, making the protein UV-absorbing. Absorption peak at 280nm, its absorbance (ie optical density value) is proportional to the protein content. In addition, the light absorbance at 238 nm of the protein solution is directly proportional to the peptide bond content. With a certain wavelength, the protein content can be determined by the direct relationship between the light absorbance of the protein solution and the protein concentration.

Ultraviolet absorption method is simple, sensitive, fast, does not consume the sample, and can be recycled after use. Low concentrations of salts such as (NH4)2SO4 commonly used in biochemical preparations and most buffers do not interfere with the assay. It is particularly suitable for rapid and continuous detection of column chromatography eluents, since at this point it is only necessary to determine the change in protein concentration without knowing its absolute value.

This method is characterized by poor accuracy of the determination of protein content, interfering substances, when using the standard curve method to determine the protein content, for those proteins with large differences in standard protein tyrosine and tryptophan content, there is a certain error. Therefore, this method is suitable for the determination of proteins similar in composition to the standard protein amino acids. If the sample contains UV-absorbing substances such as purines, pyrimidines, and nucleic acids, large interference will occur. Nucleic acid interference can be properly corrected by checking the calibration table and then calculating it. However, because the UV absorption of different proteins and nucleic acids is not the same, although the result of the measurement is still a certain amount of error.

In addition, in the UV absorption method, since the peak of protein absorption often changes due to the change of pH, it is necessary to pay attention to the pH of the solution, and the pH of the sample is determined to be consistent with the pH of the standard curve.

There are four types of ultraviolet absorption methods: light absorption at 280 nm, absorption difference at 280 nm and 260 nm, absorption difference at 215 nm and 225 nm, and peptide bond determination.

(five) Coomassie brilliant blue method (Bradford method)

(I) Experimental principle

The obvious shortcomings and many limitations of Biuret's method and Folin's method (Lowry method) have prompted scientists to find better ways to measure protein solutions.

The Bradford method established by Bradford in 1976 was based on the principle of the combination of protein and dye. This protein assay has outstanding advantages over other methods and is therefore being widely used. This method is currently the most sensitive protein assay.

Coomassie brilliant blue G-250 dye, in acidic solution and protein binding, the dye maximum absorption peak position (lmax), from 465nm to 595nm, the color of the solution from brown to blue. It is considered that dyes are mainly combined with basic amino acids (especially arginine) and aromatic amino acid residues in proteins.

The absorbance value A595 measured at 595 nm is proportional to the protein concentration.

The outstanding advantages of the Bradford method are:

(1) High sensitivity, estimated to be approximately four times higher than the Lowry method, with a minimum protein detection of 1 mg. This is because the color change produced by the combination of the protein and the dye is large, and the protein-dye complex has a higher extinction coefficient. Therefore, the light absorption value changes with the protein concentration more than the Lowry method.

(2) The assay is quick and easy, just add one reagent. It takes only about 5 minutes to complete a sample measurement. Due to the dye-protein binding process, it takes approximately 2 minutes to complete, its color can be stable within 1 hour, and the color stability is best between 5 minutes and 20 minutes. Therefore, it does not take time and strict control over time like the Lowry method.

(3) There are few interfering substances. For example, K+, Na+, Mg2+ ions, Tris buffer, sugar and sucrose, glycerol, mercaptoethanol, and EDTA that interfere with the Lowry method do not interfere with this assay.

The disadvantages of this method are:

(1) Since the contents of arginine and aromatic amino acids in various proteins are different, the Bradford method is widely used in the determination of different proteins, and g-globulins are usually used as standard proteins in the preparation of standard curves. To reduce this bias.

(2) There are still some substances that interfere with the determination of this method. The main interferences are: Detergent, Triton X-100, sodium dodecyl sulfate (SDS) and 0.1 N NaOH. (Like 0.1N acid interference Lowary method).

(3) The standard curve is also slightly non-linear and cannot be calculated using Beer's law. Instead, the standard curve can only be used to determine the concentration of an unknown protein.